Angiogenesis Pathway Gene Polymorphisms for Therapy Selection

ABSTRACT

A method for determining whether a patient in need thereof will respond to anti-VEGF antibody based chemotherapy by screening a suitable cell or tissue sample isolated from the patient for at least one genomic polymorphism or genotype selected from (i) IL-8(−251); (ii) VEGF(936); or (iii) AM (3′ CA repeats), wherein the patient is suitably treated if the corresponding genotype is (i) (T/T) for IL-8(−251); (ii) (T/T or C/T) for VEGF(936); or (iii) at least one AM allele having 14 or more 3′ CA repeats.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisionalapplication No. 60/779,018, filed Mar. 3, 2006, the contents of whichare incorporated by reference into the present disclosure.

FIELD OF THE INVENTION

This invention relates to the field of pharmacogenomics and specificallyto the application of genetic polymorphisms to diagnose and treatdiseases.

BACKGROUND OF THE INVENTION

In nature, organisms of the same species usually differ from each otherin some aspects, e.g., their appearance. The differences are geneticallydetermined and are referred to as polymorphism. Genetic polymorphism isthe occurrence in a population of two or more genetically determinedalternative phenotypes due to different alleles. Polymorphism can beobserved at the level of the whole individual (phenotype), in variantforms of proteins and blood group substances (biochemical polymorphism),morphological features of chromosomes (chromosomal polymorphism) or atthe level of DNA in differences of nucleotides (DNA polymorphism).

Polymorphism also plays a role in determining differences in anindividual's response to drugs. Cancer chemotherapy is limited by thepredisposition of specific populations to drug toxicity or poor drugresponse. Thus, for example, pharmacogenetics (the effect of geneticdifferences on drug response) has been applied in cancer chemotherapy tounderstand the significant inter-individual variations in responses andtoxicities to the administration of anti-cancer drugs, which may be dueto genetic alterations in drug metabolizing enzymes or receptorexpression. For a review of the use of germline polymorphisms inclinical oncology, see Lenz, H. -J. (2004) J. Clin. Oncol.22(13):2519-2521; Park, D. J. et al. (2006) Curr. Opin. Pharma.6(4):337-344; Zhang, W. et al. (2006) Pharma. and Genomics 16(7):475-483and U.S. Patent Publ. No. 2006/0115827. For a review of pharmacogeneticand pharmacogenomics in therapeutic antibody development for thetreatment of cancer, see Yan and Beckman (2005) Biotechniqes 39:565-568.

Polymorphism also has been linked to cancer susceptibility (oncogenes,tumor suppressor genes and genes of enzymes involved in metabolicpathways) of individuals. In patients younger than 35 years, severalmarkers for increased cancer risk have been identified. For example,prostate specific antigen (PSA) is used for the early detection ofprostate cancer in asymptomatic younger males. Cytochrome P4501A1 andgluthathione S-transferase M1 genotypes influence the risk of developingprostate cancer in younger patients. Similarly, mutations in the tumorsuppressor gene, p53, are associated with brain tumors in young adults.

Results from numerous studies suggest several genes may play a majorrole in the principal pathways of cancer progression and recurrence, andthat the corresponding germ-line polymorphisms may lead to significantdifferences at transcriptional and/or translational levels.

Moreover, while adjuvant chemotherapy and radiation lead to a noticeableimprovement in local control among those with rectal carcinoma, thechoice of optimal therapy may be compromised by a wide inter-patientvariability of treatment response and host toxicity. Since the rate ofinactivation of the administered drug compound may establish itseffectiveness in the tumor tissue, genomic variations on differentcellular mechanisms that may modify therapy efficacy may influenceefficacy. In addition, tumor microenvironment is a critical pathway incancer progression. Elements of cancer progression controlled by tumormicroenvironment genes include angiogenesis, inter-cellular adhesion,mitogenesis, and inflammation. Angiogenesis, which involves theformation of capillaries from preexisting vessels, has beencharacterized by a complex surge of events involving extensiveinterchange between cells, soluble factors (e.g. cytokines), andextracellular matrix (ECM) components (Balasubramanian (2002) Br. J.Cancer 87:1057). In addition to its fundamental role in reproduction,development, and wound repair, angiogenesis has been shown to bederegulated in cancer formation (Folkman (2002) Semin. Oncol. 29(6):15).

Improvement in the therapeutic ratio of radiation by targeting tumorcells via a combination of angiogenic blockades and radiotherapy havebeen implicated in recent studies (Gorski (1999) Cancer Res. 59:3374;Mauceri (1996) Cancer Res. 56:4311; and Mauceri (1998) Nature 394:287).However, the mechanisms by which tumor cells respond to radiationthrough these antiangiogenic/vascular agents are yet to be elucidated.Moreover, in light of the fact that oxygen is a potent radiosensitizer,cancer therapy through the combination of ionizing radiation andantiangiogenic/vascular targeting agents may seem counterintuitive sincea reduction in tumor vasculature would be expected to decrease tumorblood perfusion and lower oxygen concentration in the tumor (Wachsberger(2003) Clin. Cancer Res. 9:1957).

The interleukin family is known to play an important role in theangiogenic process. Interleukin-8, an inflammatory cytokine withangiogenic potential, has been implicated in cancer progression in avariety of cancer types including colorectal carcinoma, glioblastoma,and melanoma (Yuan (2000) Am. J. Respir. Crit. Care Med. 162:1957).Inter-cellular adhesion plays a major role in both local invasion andmetastasis. Cell adhesion molecules (CAMs), which are cell-surfaceglycoproteins that are crucial for cell-to-cell interactions, have beenshown to directly control differentiation, and interruption of normalcell-to-cell contacts has been observed in neoplastic transformation andin metastasis (Edelman (1988) Biochem. 27:3533 and Ruoslahti (1988) Ann.Rev. Biochem. 57:375). Overexpression of ICAM-1 in colorectal cancershas been shown to favor the extravasation and trafficking of cytotoxiclymphocytes toward the neoplastic cells, leading to host defense (Maurer(1998) Int. J. Cancer (Pred. Oncol.) 79:76). A polymorphism in the genecoding for Cox-2 was also studied. Cox-2 is involved in prostaglandinsynthesis, and stimulates inflammation and mitogenesis; it has beenshown to be markedly overexpressed in colorectal adenomas andadenocarcinomas when compared to normal mucosa (Eberhart (1994) Gastro.107:1183). Another family of genes playing a critical role inangiogenesis is the receptor tyrosine kinase family of fibroblast growthfactor receptors. FGFRs are also involved in tumor growth and cellmigration. The complex pathways of the tumor microenvironment havebecome the focus of widespread investigation for their role in tumorprogression.

Differences in drug metabolism, transport, signaling and cellularresponse pathways have been shown to collectively influence diversity inpatients' reactions to therapy (Evans (1999) Science 286:487).Metabolism of chemotherapeutic agents and radiation-induced products ofoxidative stress, therefore, may play a critical role in treatmentresponse. The GST superfamily participates in the detoxificationprocesses of platinum compounds (Ban (1996) Cancer Res. 56:3577 and Goto(1999) Free Rad. Res. 31:549), and was previously associated GSTP1polymorphism with response to platinum-based chemotherapy (Stoehlmacher(2002) J. Nat. Cancer Inst. 94:936).

Cell cycle regulation provides the foundation for a critical balancebetween proliferation and cell death, which are important factors incancer progression. For example, a tumor suppressor gene such as p53grants the injured cell time to repair its damaged DNA by inducing cellcycle arrest before reinitiating replicative DNA synthesis and/ormitosis (Kastan (1991) Cancer Res. 51:6304). More importantly, when p53is activated based on DNA damage or other activating factors, it caninitiate downstream events leading to apoptosis (Levine (1992) N. Engl.J. Med. 326:1350). The advent of tumor recurrence after radiationtherapy depends significantly on how the cell responds to the inducedDNA damage; that is, increased p53 function should induce apoptosis inthe irradiated cell and thereby prevent proliferation of cancerouscells, whereas decreased p53 function may decrease apoptotic rates.

Finally, DNA repair capacity contributes significantly to the cell'sresponse to chemoradiation treatment (Yanagisawa (1998) Oral Oncol.34:524). Patient variability in sensitivity to radiotherapy can beattributed to either the amount of damage induced upon radiationexposure or the cell's ability to tolerate and repair the damage (Nunez(1996) Rad. Onc. 39:155). Irradiation can damage DNA directly, orindirectly via reactive oxygen species, and the cell has severalpathways to repair DNA damage including double-stranded break repair(DSBR), nucleotide excision repair (NER), and base excision repair(BER). An increased ability to repair direct and indirect damage causedby radiation will inherently lower treatment capability and hence maylead to an increase in tumor recurrence.

DESCRIPTION OF THE EMBODIMENTS

This invention provides methods to detect polymorphisms that have beendetermined to be clinically relevant to cancers that have beensuccessfully treated with anti-VEGF antibody treatments.

In one aspect, the method requires determining the presence or absenceof at least one allelic variant of a predetermined gene selected fromthe group consisting of a polymorphism at nucleotide −251 in theinterleukin 8 gene [8(−251)], a polymorphism at nucleotide 936 of thevascular endothelial growth factor gene [VEGF(936)] and a polymorphismin the 3′ end of the adrenomedullin gene [AM (3′ CA repeat)]. In anotheraspect, it requires determining two or more of the above-notedpolymorphic regions. In a further aspect, it requires identifying allthree. The genes of interest are selected from those shown to beinvolved with colon, breast, lung or ovarian cancer.

The invention also provides the tools to execute the methods of thisinvention. In one aspect, the tools can include using nucleic acidsencompassing the polymorphic region of interest or adjacent to thepolymorphic region as probes or primers.

In one aspect, the sample to be tested is the actual tumor tissue. Inanother aspect the sample can be normal tissue isolated adjacent to thetumor. In yet a further aspect, the sample is normal tissuecorresponding to the tumor tissue (e.g., normal ovarian tissue when thecancer is ovarian cancer). In a further aspect, the sample is any tissueof the patient, and can include peripheral blood lymphocytes.

In another aspect, the invention comprises administration of anappropriate therapy or combination therapy after identification of theclinical correlation of the polymorphism of interest.

In yet a further embodiment, the invention provides a kit for amplifyingand/or for determining the molecular structure of at least a portion ofthe gene of interest, comprising a probe or primer capable of detectingto the gene of interest and instructions for use. In one embodiment, theprobe or primer is capable of detecting an allelic variant of the geneof interest.

It will be appreciated by one of skill in the art that the embodimentssummarized above may be used together in any suitable combination togenerate additional embodiments not expressly recited above, and thatsuch embodiments are considered to be part of the present invention.

MODES FOR CARRYING OUT THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature for example in the followingpublications. See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(F. M. Ausubel et al. eds. (1987)); the series METHODS IN ENZYMOLOGY(Academic Press, Inc., N.Y.); PCR: A PRACTICAL APPROACH (M. MacPhersonet al. IRL Press at Oxford University Press (1991)); PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995));ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane eds. (1988)); ANIMALCELL CULTURE (R. I. Freshney ed. (1987)); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed. (1984)); Mullis et al. U.S. Pat. No. 4,683,195; NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. (1984)); TRANSCRIPTIONAND TRANSLATION (B. D. Hames & S. J. Higgins eds. (1984)): IMMOBILIZEDCELLS AND ENZYMES (IRL Press (1986)); B. Perbal, A PRACTICAL GUIDE TOMOLECULAR CLONING (1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J.H. Miller and M. P. Calos eds. (1987) Cold Spring Harbor Laboratory);IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and Walker,eds., Academic Press, London (1987)); HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Volumes I-1V (D. M. Weir and C. C. Blackwell, eds. (1986)):MANIPULATING THE MOUSE EMBRYO (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1986)).

Definitions

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The term “recombinant protein” refers to a polypeptide which is producedby recombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extrachromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

The term “allelic variant of a polymorphic region of the gene ofinterest” refers to a region of the gene of interest having one of aplurality of nucleotide sequences found in that region of the gene inother individuals.

The expression “amplification of polynucleotides” includes methods suchas PCR, ligation amplification (or ligase chain reaction, LCR) andamplification methods. These methods are known and widely practiced inthe art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis etal., 1990 (for PCR); and Wu, D. Y. et al. (1989) Genomics 4:560-569 (forLCR). In general, the PCR procedure describes a method of geneamplification which is comprised of (i) sequence-specific hybridizationof primers to specific genes within a DNA sample (or library), (ii)subsequent amplification involving multiple rounds of annealing,elongation, and denaturation using a DNA polymerase, and (iii) screeningthe PCR products for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the genomic locus to beamplified.

Reagents and hardware for conducting PCR are commercially available.Primers useful to amplify sequences from a particular gene region arepreferably complementary to, and hybridize specifically to sequences inthe target region or its flanking regions. Nucleic acid sequencesgenerated by amplification may be sequenced directly. Alternatively theamplified sequence(s) may be cloned prior to sequence analysis. A methodfor the direct cloning and sequence analysis of enzymatically amplifiedgenomic segments is known in the art.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

The term “genotype” refers to the specific allelic composition of anentire cell or a certain gene, whereas the term “phenotype' refers tothe detectable outward manifestations of a specific genotype.

As used herein, the term “gene' or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame and including atleast one exon and (optionally) an intron sequence. The term “intron”refers to a DNA sequence present in a given gene which is spliced outduring mRNA maturation.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous that position. A degree of homology between sequences isa function of the number of matching or homologous positions shared bythe sequences. An “unrelated” or “non-homologous” sequence shares lessthan 40% identity, though preferably less than 25% identity, with one ofthe sequences of the present invention.

The term “a homolog of a nucleic acid” refers to a nucleic acid having anucleotide sequence having a certain degree of homology with thenucleotide stranded nucleic acid is intended to include nucleic acidshaving a nucleotide sequence which has a certain degree of homology withor with the complement thereof. In one aspect, homologs of nucleic acidsare capable of hybridizing to the nucleic acid or complement thereof.

The term “interact' as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a hybridization assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may be,for example, protein-protein, protein-nucleic acid, protein-smallmolecule or small molecule-nucleic acid in nature.

The term “isolated” as used herein with respect to a patient samplerefers to tissue, cells, genetic material and nucleic acids, such as DNAor RNA, separated from other cells or tissue or DNAs or RNAs,respectively, that are present in the natural source. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,derivatives, variants and analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or antisense) and double-strandedpolynucleotides. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes ofclarity, when referring herein to a nucleotide of a nucleic acid, whichcan be DNA or an 'RNA, the terms “adenosine”, “cytidine”, “guanosine”,and thymidine” are used. It is understood that if the nucleic acid isRNA, a nucleotide having a uracil base is uridine.

The terms “oligonucleotide” or “polynucleotide”, or “portion,” or“segment” thereof refer to a stretch of polynucleotide residues which islong enough to use in PCR or various hybridization procedures toidentify or amplify identical or related parts of mRNA or DNA molecules.The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion thereof. A portion of a gene of which there are atleast two different forms, i.e., two different nucleotide sequences, isreferred to as a “polymorphic region of a gene”. A polymorphic regioncan be a single nucleotide, the identity of which differs in differentalleles.

A “polymorphic gene” refers to a gene having at least one polymorphicregion.

As used herein, the term “gene of interest” or “polymorphism ofinterest” intends those exemplified herein, e.g., VEGF(936), IL-8(−251)and AM (3′ CA repeats).

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.For example, in the case of cancer, treatment includes a reduction incachexia, increase in survival time, elongation in time to tumorprogression, reduction in tumor mass, reduction in tumor burden and/or aprolongation in time to tumor metastasis, each as measured by standardsset by the National Cancer Institute and the U.S. Food and DrugAdministration for the approval of new drugs. See Johnson et al. (2003)J. Clin. Oncol. 21(7):1404-1411.

A “response” shall mean a 50% reduction in tumor.

A “complete response” (CR) to a therapy defines patients with evaluablebut non-measurable disease, whose tumor and all evidence of disease haddisappeared.

A “partial response” (PR) to a therapy defines patients with anythingless than complete response were simply categorized as demonstratingpartial response.

“Non-response” (NR) to a therapy defines patients whose evidence ofdisease has remained constant or has progressed.

“Time to tumor progression” is the time between treatment and initialresponse and the time when resistance to initial treatment or loss oftreatment efficacy.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages. Such delivery is dependent ona number of variables including the time period for which the individualdosage unit is to be used, the bioavailability of the therapeutic agent,the route of administration, etc. It is understood, however, thatspecific dose levels of the therapeutic agents of the present inventionfor any particular subject depends upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, and diet of the subject, the time ofadministration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form ofadministration. Treatment dosages generally may be titrated to optimizesafety and efficacy. Typically, dosage-effect relationships from invitro and/or in vivo tests initially can provide useful guidance on theproper doses for patient administration. In general, one will desire toadminister an amount of the antibody or compound that is effective toachieve a serum level commensurate with the concentrations found to beeffective in vitro. Determination of these parameters is well within theskill of the art. These considerations, as well as effectiveformulations and administration procedures are well known in the art andare described in standard textbooks. Consistent with this definition, asused herein, the term “therapeutically effective amount” is an amountsufficient to treat a the cancer.

The compositions and compounds can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternalinjection or infusion, subcutaneous injection, or implant), byinhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g.,urethral suppository) or topical routes of administration (e.g., gel,ointment, cream, aerosol, etc.) and can be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, excipients,and vehicles appropriate for each route of administration. The inventionis not limited by the route of administration, the formulation or dosingschedule.

Despite advances in chemotherapy, ovarian cancer remains a major causeof cancer mortality worldwide. It has therefore become essential toidentify novel therapeutic targets, such as angiogenesis, which is acomplex process regulated by the delicate balance between various localpro-angiogenic and anti-angiogenic proteins. Bevacizumab is a monoclonalantibody that binds to VEGF, has shown significant activity in colon,breast, lung and ovarian cancer. It is sold under the tradename Avastin(Genentech). The antibody targets VEGF, a protein made by cells thatstimulates the production of new blood vessels. VEGF is structurallyrelated to platelet-derived growth factor (PDGF). The gene is located onchromosome 6p12.

The key enzymes of the VEGF pathway are: Vascular Endothelial GrowthFactor (VEGF), VEGF Receptor (VEGFR), Hypoxia Inducible Factor-1 (HIF αand β-subunit), Neuropilin-1 (NRP), Interleukin-8 (IL-8), Adrenomedullin(AM) and Leptin. Unexpectedly, Applicant found that IL-8(−251) is amolecular marker for response to Bevacizumab based cancer therapy.Patients homozygous (T/T) at nucleotide −251 for the IL-8 gene inpatient samples were more responsive to this therapy than patientsheterozygous (A/T) or homozygous (A/A). In this study, responsivenesswas measured a 50% reduction in tumor. Patients with any T (T/T or C/T)at nucleotide 936 for the VEGF gene showed more than double time totumor progression than patients that were (C/C) at the same locus.Patients carrying one AM allele with more than 14 CA repeats were betterresponders and patients with both AM 3′ end alleles having 14 or more CArepeats had the longest time to progression. In a further aspect, the AMgenotype contains one allele containing 14 3′ CA repeats oralternatively, both alleles contain 14 3′ CA repeats.

Thus, a method is described to identify patients in need thereof,suitably treated by Bevacizumab based chemotherapy by screening orassaying a suitable patient sample for a polymorphism that has beencorrelated by Applicant to positive therapeutic response or separately,to time to tumor progression. Although Bevacizumab was the antibodytherapy that was administered to patients in this reported study, theresults can be extrapolated to any therapy which includes an anti-VEGFantibody with the same or similar activity to Bevacizumab. Antibodieswith the same or similar activity can be described as “biologicallyequivalent”. Methods to make and identify such antibodies are describedherein.

In one aspect, the anti-VEGF antibody binds to any region or epitope onVEGF. In an alternative embodiment, the antibody binds to the sameepitope on VEGF as Bevacizumab. In a further aspect, the antibody is avariant or derivative of Bevacizumab of any of the above and can includefunction fragments, derivatives or such antibodies conjugated to otheragents.

In addition, although the particular patient population was undergoingtreatment for ovarian cancer, the method is useful for any cancer thatis amendable to anti-VEGF based chemotherapy as defined above. Suchpatients include, but are not limited to colon cancer patients, breastcancer patients and lung cancer patients, in addition to the exemplifiedovarian cancer patients.

Suitable patient samples include tumor samples, normal tissuecorresponding to the tumor type, tissue adjacent to the tumor andperipheral blood lymphocytes.

In one aspect a method is described for determining whether a patient inneed thereof will respond to anti-VEGF antibody based chemotherapy byscreening a suitable cell or tissue sample isolated from said patientfor at least one genomic polymorphism or genotype selected from (i)IL-8(−251); (ii) VEGF(936); or (iii) AM (3′ CA repeats), wherein thepatient is suitably treated if the corresponding genotype is (i) (T/T)for IL-8(−251); (ii) (T/T or C/T) for VEGF(936); or (iii) at least oneallele having 14 or more CA repeats (AM 3′). Patients carrying one AMallele with more than 14 CA repeats were better responders and patientswith both AM 3′ end alleles having 14 or more CA repeats were the bestresponse to the therapy. In a further aspect, the patient is selectedand considered a candidate for therapy when his or her AM genotypecontains one allele containing 14 3′ CA repeats or alternatively, bothalleles contain 14 3′ CA repeats.

In one aspect, the anti-VEGF antibody based therapy comprises theadministration of a Bevacizumab antibody or its biological equivalent.In a further aspect, the patient is a cancer patient suffering fromovarian cancer, lung cancer, breast cancer or colon cancer. In anotheraspect, the cancer treatment further comprises administration of aneffective amount of cyclophosphamide, e.g., a very low dose ofcyclophosphamide. Effective amounts vary with the patient, the cancerbeing treated and can be determined by the treating physician.

Further described is a method for selecting anti-VEGF antibody basedchemotherapy for a patient in need thereof by screening a suitable cellor tissue sample isolated from said patient for at least one genomicpolymorphism or genotype selected from (i) IL-8(−251); (ii) VEGF(936);or (iii) AM (3′ CA repeats), wherein the therapy is selected if thepatient's corresponding genotype is (i) (T/T) for IL-8(−251); (ii) (T/Tor C/T) for VEGF(936); or (iii) and one or both alleles having 14 ormore CA repeats (AM 3′). In one aspect the patient is positive for twoof the three genotypes. In a further aspect, the patient is positive forIL-8 (−251) and VEGF (936) and has one AM allele with 14 or more (3′) CArepeats. In yet a further aspect, the patient is positive for IL-8(−251) and VEGF (936) and has two AM alleles with 14 or more (3′) CArepeats.

In one aspect, the anti-VEGF antibody based therapy comprises theadministration of an effective amount of Bevacizumab antibody or itsbiological equivalent. In a further aspect, the patient is a cancerpatient suffering from a cancer of the group ovarian cancer, lungcancer, breast cancer and colon cancer. In a yet further aspect, thecancer treatment further comprises administration of an effective amountof cyclophosphamide, e.g., a very lose dose of cyclophosphamide.Effective amounts vary with the patient, the cancer being treated andcan be determined by the treating physician.

In general, a therapy is considered to “treat” cancer if it provides oneor more of the following treatment outcomes: reduce or delay recurrenceof the cancer after the initial therapy (i.e., time to tumorprogression; reduction in tumor mass; reduction in tumor burden;increase median survival time or a decrease in tumor metastases). Themethod is particularly suited to determining which patients will beresponsive or experience a positive treatment outcome to achemotherapeutic regimen involving administration of Bevacizumab basedchemotherapy or an equivalent anti-VEGF antibody therapy.

In one embodiment, the chemotherapeutic regimen further comprisesradiation therapy or other adjuvant chemotherapy such as administrationof a low dose of cyclophoshamide.

In addition to selecting a treatment protocol for the patient in needthereof, the method enables a doctor: 1) to more effectively prescribe adrug that will address the molecular basis of the disease or condition;2) to better determine the appropriate dosage of a particular drug and3) to identify novel targets for drug development.

Identification of the polymorphism can be accomplished by molecularcloning of the specified allele and subsequent sequencing of that alleleusing techniques known in the art. Alternatively, the gene sequences canbe amplified directly from a genomic DNA preparation from the tumortissue using PCR, and the sequence composition is determined from theamplified product. As described more fully below, numerous methods areavailable for analyzing a subject's DNA for mutations at a given geneticlocus such as the gene of interest.

The polymorphism of interest can be identified using the methodsexemplified below or any other of the various methods known to thoseskilled in the art. Art known methods include without limitation DNAmicroarray technology that also has many varieties, e.g., DNA chipdevices and systems high-density microarrays for high-throughputscreening applications and lower-density microarrays. Methods formicroarray fabrication are known in the art and include various inkjetand microjet deposition or spotting technologies and processes, in situor on-chip photolithographic oligonucleotide synthesis processes, andelectronic DNA probe addressing processes. The DNA microarrayhybridization applications has been successfully applied in the areas ofgene expression analysis and genotyping for point mutations, singlenucleotide polymorphisms (SNPs), and short tandem repeats (STRs).Additional methods include interference RNA microarrays and combinationsof microarrays and other methods such as laser capture microdisection(LCM), comparative genomic hybridization (CGH) and chromatinimmunoprecipitation (ChiP). For a review of these technologies, see Heet al. (2007) Adv. Exp. Med. Biol. 593:117-133 and Heller (2002) AnnuRev. Biomed. Eng. 4:129-153.

Other art-known methods include, without limitation PCR, xMAP, invaderassay, mass spectrometry, and pyrosequencing (Wang et al. (2007)593:105-106). The patent literature also describes art known methods forSNP analysis.

A detection method is allele specific hybridization using probesoverlapping the polymorphic site and having about 5, or alternatively10, or alternatively 20, or alternatively 25, or alternatively 30nucleotides around the polymorphic region. In another embodiment of theinvention, several probes capable of hybridizing specifically to theallelic variant are attached to a solid phase support, e.g., a “chip”.Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. For example a chip can hold up to250,000 oligonucleotides (GeneChip, Affymetrix). Mutation detectionanalysis using these chips comprising oligonucleotides, also termed “DNAprobe arrays” is described e.g., in Cronin et al. (1996) Human Mutation7:244.

In other detection methods, it is necessary to first amplify at least aportion of the gene of interest prior to identifying the allelicvariant. Amplification can be performed, e.g., by PCR and/or LCR,according to methods known in the art. In one embodiment, genomic DNA ofa cell is exposed to two PCR primers and amplification for a number ofcycles sufficient to produce the required amount of amplified DNA.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known to those of skill in the art.These detection schemes are useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of the gene ofinterest and detect allelic variants, e.g., mutations, by comparing thesequence of the sample sequence with the corresponding wild-type(control) sequence. Exemplary sequencing reactions include those basedon techniques developed by Maxam and Gilbert ((1997) Proc. Natl Aced Sd,USA 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci,74:5463). It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the subject assays(Biotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example, U.S. Pat. No. 5,547,835 and International PatentApplication Publication Number WO94/16101, entitled DNA Sequencing byMass Spectrometry by H. Koster; U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/21822 entitled“DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation” by H.Koster; U.S. Pat. No. 5,605,798 and International Patent Application No.PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H.Koster; Cohen et al. (1996) Adv. Chromat. 36:127-162; and Griffin et al.(1993) Appl. Biochem Bio. 38:147-159). It will be evident to one skilledin the art that, for certain embodiments, the occurrence of only one,two or three of the nucleic acid bases need be determined in thesequencing reaction. For instance, A-track or the like, e.g., where onlyone nucleotide is detected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA Sequencing Employing A MixedDNA-Polymer Chain Probe” and U.S. Pat. No. 5,571,676 entitled “MethodFor Mismatch-Directed In Vitro DNA Sequencing.”

In some cases, the presence of the specific allele in DNA from a subjectcan be shown by restriction enzyme analysis. For example, the specificnucleotide polymorphism can result in a nucleotide sequence comprising arestriction site which is absent from the nucleotide sequence of anotherallelic variant.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (see, e.g., Myers et al. (1985) Science 230:1242). Ingeneral, the technique of “mismatch cleavage” starts by providingheteroduplexes formed by hybridizing a control nucleic acid, which isoptionally labeled, e.g., RNA or DNA, comprising a nucleotide sequenceof the allelic variant of the gene of interest with a sample nucleicacid, e.g., RNA or DNA, obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as duplexes formed based onbasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine whether the control and sample nucleicacids have an identical nucleotide sequence or in which nucleotides theyare different. See, for example, U.S. Pat. No. 6,455,249, Cotton et al.(1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzy. 217:286-295. In another embodiment, the control or sample nucleicacid is labeled for detection.

In other embodiments, alterations in electrophoretic mobility is used toidentify the particular allelic variant. For example, single strandconformation polymorphism (SSCP) may be used to defect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sd USA 86:2766; Cotton (1993)Mutat. Res. 285:125-144 and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control nucleicacids are denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet.7:5).

In yet another embodiment, the identity of the allelic variant isobtained by analyzing the movement of a nucleic acid comprising thepolymorphic region in polyacrylamide gels containing a gradient ofdenaturant, which is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGEis used as the method of analysis, DNA will be modified to insure thatit does not completely denature, for example by adding a GC clamp ofapproximately 40 by of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between 2 nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl.Acad. Sci USA 86:6230 and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the detection of the nucleotide changes in thepolylmorphic region of the gene of interest. For example,oligonucleotides having the nucleotide sequence of the specific allelicvariant are attached to a hybridizing membrane and this membrane is thenhybridized with labeled sample nucleic acid. Analysis of thehybridization signal will then reveal the identity of the nucleotides ofthe sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the allelic variant of interest in the center of the molecule(so that amplification depends on differential hybridization) (Gibbs etal. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end ofone primer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238 and Newtonet al. (1989) Nucl. Acids Res. 17:2503). This technique is also termed“PROBE” for Probe Oligo Base. Extension. In addition it may be desirableto introduce a novel restriction site in the region of the mutation tocreate cleavage-based detection (Gasparini et al. (1992) Mol. CellProbes 6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Laridegren, U. et al. Science241:1077-1 080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled, If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect the specific allelic variant of the polymorphic regionof the gene of interest. For example, U.S. Pat. No. 5,593,826 disclosesan OLA using an oligonucleotide having 3′-amino group and a5′-phosphorylated oligonucleotide to form a conjugate having aphosphoramidate linkage. In another variation of OLA described in Tobeet al. (1996) Nucleic Acids Res. 24: 3728), OLA combined with PCRpermits typing of two alleles in a single microtiter well. By markingeach of the allele-specific primers with a unique hapten, i.e.digoxigenin and fluorescein, each OLA reaction can be detected by usinghapten specific antibodies that are labeled with different enzymereporters, alkaline phosphatase or horseradish peroxidase. This systempermits the detection of the two alleles using a high throughput formatthat leads to the production of two different colors.

The invention further provides methods for detecting the singlenucleotide polymorphism in the gene of interest. Because singlenucleotide polymorphisms in the gene of interest. Because singlenucleotide polymorphisms constitute sites of variation flanked byregions of invariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation and it is unnecessary to determine a complete genesequence for each patient. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorpbration renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of the polymorphic site(Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087)). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). This methoduses mixtures of labeled terminators and a primer that is complementaryto the sequence 3′ to a polymorphic site. The labeled terminator that isincorporated is thus determined by, and complementary to, the nucleotidepresent in the polymorphic site of the target molecule being evaluated.In contrast to the method of Cohen et al. (French Patent 2,650,840; PCTAppln. No. WO91/02087) the method of Goelet, P. et al. supra, ispreferably a heterogeneous phase assay, in which the primer or thetarget molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P. (1990) Nucl.Acids Res. 18:3671; Syvanen, A. -C., et al. (1990) Genomics 8:684-692;Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1:159-164;Ugozzoli, L. et al. (1992) GATA 9:107-112; Nyren, P. et al. (1993) Anal.Biochem. 208:171-175). These methods differ from GBA™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A. -C.,et al. (1993) Amer. J. Hum. Genet. 52:46-59).

If the polymorphic region is located in the coding region of the gene ofinterest, yet other methods than those described above can be used fordetermining the identity of the allelic variant. For example,identification of the allelic variant, which encodes a mutated signalpeptide, can be performed by using an antibody specifically recognizingthe mutant protein in, e.g., immunohistochemistry orimmunoprecipitation. Antibodies to the wild-type or signal peptidemutated forms of the signal peptide proteins can be prepared accordingto methods known in the art.

Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits, such as those described below, comprisingat least one probe or primer nucleic acid described herein, which may beconveniently used, e.g., to determine whether a subject has or is atrisk of developing disease such as colorectal cancer.

Sample nucleic acid for use in the above-described diagnostic andprognostic methods can be obtained from any cell type or tissue of asubject. For example, a subject's bodily fluid (e.g. blood) can beobtained by known techniques (e.g., venipuncture). Alternatively,nucleic acid tests can be performed on dry samples (e.g., hair or skin).Fetal nucleic acid samples can be obtained from maternal blood asdescribed in International Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi can be obtained forperforming prenatal testing.

Diagnostic procedures can also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents can be used as probes and/or primers for such insitu procedures for (see, for example, Nuovo, G. J. (1992) “PCR In SituHybridization: Protocols And Applications”, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles can also be assessed in such detectionschemes. Fingerprint profiles can be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

The invention described herein relates to methods and compositions fordetermining and identifying the allele present at the gene of interest'slocus. This information is useful to diagnose and prognose diseaseprogression as well as select the most effective treatment amongtreatment options. Probes can be used to directly determine the genotypeof the sample or can be used simultaneously with or subsequent toamplification. The term “probes” includes naturally occurring orrecombinant single- or double-stranded nucleic acids or chemicallysynthesized nucleic acids. They may be labeled by nick translation,Klenow fill-in reaction, PCR or other methods known in the art. Probesof the present invention, their preparation and/or labeling aredescribed in Sambrook et al. (1989) supra. A probe can be apolynucleotide of any length suitable for selective hybridization to anucleic acid containing a polymorphic region of the invention. Length ofthe probe used will depend, in part, on the nature of the assay used andthe hybridization conditions employed.

In one embodiment of the invention, probes are labeled with twofluorescent dye molecules to form so-called “molecular beacons” (Tyagi,S. and Kramer, F. R. (1996) Nat. Biotechnol. 14:303-8). Such molecularbeacons signal binding to a complementary nucleic acid sequence throughrelief of intramolecular fluorescence quenching between dyes bound toopposing ends on an oligonucleotide probe. The use of molecular beaconsfor genotyping has been described (Kostrikis, L. G. (1998) Science279:1228-9) as has the use of multiple beacons simultaneously (Marras,S. A. (1999) Genet. Anal. 14:151-6). A quenching molecule is useful witha particular fluorophore if it has sufficient spectral overlap tosubstantially inhibit fluorescence of the fluorophore when the two areheld proixmal to one another, such as in a molecular beacon, or whenattached to the ends of an oligonucleotide probe from about 1 to about25 nucleotides.

Labeled probes also can be used in conjunction with amplification of apolymorphism. (Holland et al. (1991) Proc. Natl. Acad. Sci.88:7276-7280). U.S. Pat. No. 5,210,015 by Gelfand et al. describefluorescence-based approaches to provide real time measurements ofamplification products during PCR. Such approaches have either employedintercalating dyes (such as ethidium bromide) to indicate the amount ofdouble-stranded DNA present, or they have employed probes containingfluorescence-quencher pairs (also referred to as the “Taq-Man” approach)where the probe is cleaved during amplification to release a fluorescentmolecule whose concentration is proportional to the amount ofdouble-stranded DNA present. During amplification, the probe is digestedby the nuclease activity of a polymerase when hybridized to the targetsequence to cause the fluorescent molecule to be separated from thequencher molecule, thereby causing fluorescence from the reportermolecule to appear. The Taq-Man approach uses a probe containing areporter molecule—quencher molecule pair that specifically anneals to aregion of a target polynucleotide containing the poymorphism.

Probes can be affixed to surfaces for use as “gene chips.” Such genechips can be used to detect genetic variations by a number of techniquesknown to one of skill in the art. In one technique, oligonucleotides arearrayed on a gene chip for determining the DNA sequence of a by thesequencing by hybridization approach, such as that outlined in U.S. Pat.Nos. 6,025,136 and 6,018,041. The probes of the invention also can beused for fluorescent detection of a genetic sequence. Such techniqueshave been described, for example, in U.S. Pat. Nos. 5,968,740 and5,858,659. A probe also can be affixed to an electrode surface for theelectrochemical detection of nucleic acid sequences such as described byKayyem et al. U.S. Pat. No. 5,952,172 and by Kelley, S. O. et al. (1999)Nucleic Acids Res. 27:4830-4837.

Additionally, the isolated nucleic acids used as probes or primers maybe modified to become more stable. Exemplary nucleic acid moleculeswhich are modified include phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564 and 5,256,775).

Methods of Treatment

The invention further provides methods of treating subjects havingcancer after they have been identified as suitable for anti-VEGF basedchemotherapy by administering an effective amount of an anti-VEGFantibody. In one embodiment, the method comprises (a) determining theidentity of the allelic variant identified herein as relevant tosensitivity to VEGF-antibody based chemotherapy; and (b) administeringto the subject an effective amount of an anti-VEGF antibody or anequivalent of the antibody.

The antibody administered to the patient can be an anti-VEGF monoclonalantibody, an anti-VEGF polyclonal antibody, an active fragment thereofor a derivative or a variant thereof. These compositions can beconjugated to additional diagnostic or therapeutic agents. They can beadministered alone or in combination with other effective orexperimental therapies. Antibodies with the same or similar affinity andbiological activity to Bevacizumab are examples of such antibodies.

Typically, the antibodies for use in these methods are monoclonalantibodies, although in certain aspects, polyclonal antibodies can beutilized. They can be chimeric, humanized, or totally human. Theantibodies can be produced in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes,etc. So long as the fragment or derivative retains specificity ofbinding or neutralization ability as the antibodies of this invention itcan be used. Antibodies can be tested for specificity of binding bycomparing binding to appropriate antigen to binding to irrelevantantigen or antigen mixture under a given set of conditions. If theantibody binds to the appropriate antigen at least 2, 5, 7, andpreferably 10 times more than to irrelevant antigen or antigen mixturethen it is considered to be specific.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oljgonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) Biolnvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu etal., (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998)Immunol. 93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al.,(1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al., Proc. Natl. Acad. Sci. USA (1996)93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134 (1994)

Antibody variants or biological equivalents thereof can also be preparedby delivering a polynucleotide encoding an antibody of this invention toa suitable host such as to provide transgenic animals or mammals, suchas goats, cows, horses, sheep, and the like, that produce suchantibodies in their milk. These methods are known in the art and aredescribed for example in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316;5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody variants also can be prepared by delivering a polynucleotide ofthis invention to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbol. Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies of the presentinvention can also be produced using transgenic plants, according toknow methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally part or all of the non-human orhuman CDR sequences are maintained while the non-human sequences of thevariable and constant regions are replaced with human or other aminoacids.

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering ofantibodies of the present invention can be performed using any knownmethod, such as but not limited to those described in U.S. Pat. Nos.5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; and 4,816,567.

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel, N. D. et al. (2000) Infection andImmunity April 2000:1820-1826; Gallo, M. L. et al. (2000) European J. ofImmun. 30:534-540; Green, L. L. (1999) J. of Immun. Methods 231:11-23;Yang, X -D et al. (1999A) J. of Leukocyte Biology 66:401-410; Yang, X -D(1999B) Cancer Research 59(6):1236-1243; Jakobovits, A. (1998) AdvancedDrug Delivery Reviews 31:33-42; Green, L. and Jakobovits, A. (1998) J.Exp. Med. 188(3):483-495; Jakobovits, A. (1998) Exp. Opin. Invest. Drugs7(4):607-614; Tsuda, H. et al. (1997) Genomics 42:413-421; Sherman-Gold,R. (1997). Genetic Engineering News 17(14); Mendez, M. et al. (1997)Nature Genetics 15:146-156; Jakobovits; A. (1996) Weir's Handbook ofExperimental Immunology, The Integrated Immune System Vol. IV,194.1-194.7; Jakobovits, A. (1995) Current Opinion in Biotechnology6:561-566; Mendez, M. et al. (1995) Genomics 26:294-307; Jakobovits, A.(1994) Current Biology 4(8):761-763; Arbones, M. et al. (1994) Immunity1(4):247-260; Jakobovits, A. (1993) Nature 362(6417):255-258;Jakobovits, A. et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555;Kucherlapati, et al. U.S. Pat. No. 6,075,181.)

Human monoclonal antibodies can also be produced by a hybridoma whichincludes a B cell obtained from a transgenic nonhuman animal, e.g., atransgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The antibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (SI) connected to a light chainvariable domain (V) in the same polypeptide chain (VH V). (See forexample, EP 404,097; WO 93/11161; and Hollinger et al., (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448.) By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (See also, U.S. Pat. No. 6,632,926 toChen et al. which discloses antibody variants that have one or moreamino acids inserted into a hypervariable region of the parent antibodyand a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for theantigen.)

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making the is known in the art and described in Zapata etal. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodiescomprise a pair of tandem Fd segments (V-C 1-VH-C1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The antibodies of this invention can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells, or alternatively from aprokaryotic cells as described above.

Antibodies can also be conjugated, for example, to a pharmaceuticalagent, such as chemotherapeutic drug or a toxin. They can be linked to acytokine, to a ligand, to another antibody. Suitable agents for couplingto antibodies to achieve an anti-tumor effect include cytokines, such asinterleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers,for use in photodynamic therapy, including aluminum (III) phthalocyaninetetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, suchas iodine-131 (¹³¹I), yttrium-90 (⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213(²¹³Bi), technetium-99m (^(99m)Tc), rhenium-186 (¹⁸⁶Re), and rhenium-188(¹⁸⁸Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinesecobra (naja naja atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestriction (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; ly207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

Modified antibodies of the invention also can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis. Seegenerally, Hermanson, G. T., BIOCONJUGATE TECHNIQUES, Academic Press:San Diego, Calif. (1996).

Kits

As set forth herein, the invention also provides diagnostic methods fordetermining the type of allelic variant of a polymorphic region presentin the gene of interest or the expression level of a gene of interest.In some embodiments, the methods use probes or primers comprisingnucleotide sequences which are complementary to the polymorphic regionof the gene of interest. Accordingly, the invention provides kits forperforming these methods.

In an embodiment, the invention provides a kit for determining whether asubject is likely to respond to anti-VEGF antibody based chemotherapy.The kits contain one of more of the compositions described above andinstructions for use. As an example only, the invention also provideskits for determining response to cancer treatment containing a first andsecond oligonucleotide specific for the polymorphic region of the geneof interest, e.g., IL-8 (251), VEGF(936) or AM (3′ CA repeat).Oligonucleotides “specific for” a genetic locus bind either to thepolymorphic region of the locus or bind adjacent to the polymorphicregion of the locus. For oligonucleotides that are to be used as primersfor amplification, primers are adjacent if they are sufficiently closeto be used to produce a polynucleotide comprising the polymorphicregion. In one embodiment, oligonucleotides are adjacent if they bindwithin about 1-2 kb, and preferably less than 1 kb from thepolymorphism. Specific oligonucleotides are capable of hybridizing to asequence, and under suitable conditions will not bind to a sequencediffering by a single nucleotide.

The kit can comprise at least one probe or primer which is capable ofspecifically hybridizing to the polymorphic region of the gene ofinterest and instructions for use. The kits preferably comprise at leastone of the above described nucleic acids. Preferred kits for amplifyingat least a portion of the gene of interest comprise two primers, atleast one of which is capable of hybridizing to the allelic variantsequence. Such kits are suitable for detection of genotype by, forexample, fluorescence detection, by electrochemical detection, or byother detection.

Oligonucleotides, whether used as probes or primers, contained in a kitcan be detectably labeled. Labels can be detected either directly, forexample for fluorescent labels, or indirectly. Indirect detection caninclude any detection method known to one of skill in the art, includingbiotin-avidin interactions, antibody binding and the like. Fluorescentlylabeled oligonucleotides also can contain a quenching molecule.Oligonucleotides can be bound to a surface. In one embodiment, thepreferred surface is silica or glass. In another embodiment, the surfaceis a metal electrode.

Yet other kits of the invention comprise at least one reagent necessaryto perform the assay. For example, the kit can comprise an enzyme.Alternatively the kit can comprise a buffer or any other necessaryreagent.

Conditions for incubating a nucleic acid probe with a test sample dependon the format employed in the assay, the detection methods used, and thetype and nature of the nucleic acid probe used in the assay.

The kits can include all or some of the positive controls, negativecontrols, reagents, primers, sequencing markers, probes and antibodiesdescribed herein for determining the subject's genotype in thepolymorphic region of the gene of interest.

As amenable, these suggested kit components may be packaged in a mannercustomary for use by those of skill in the art. For example, thesesuggested kit components may be provided in solution or as a liquiddispersion or the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXPERIMENTAL EXAMPLE

IL-8 also has been identified as being involved in the EGFR pathway. Acommon single nucleotide polymorphism was identified 251 base pairsupstream of the IL-8 transcription start site and the IL-8 (−251A)allele was found to be associated with increased IL-8 production.

The VEGF (C936T) gene polymorphism, located in the 3′-UTR of VEGF gene,has been associated with low VEGF plasma levels and reduced breastcancer risk.

Adrenomedullin (AM) is a hypotensive peptide widely produced in thecardiovascular organs and tissues such as the heart, kidney and vascularcells. The 3′-end of the gene is flanked by a microsatellite marker ofcytosine adenine (CA) repeats. In Japanese, four types of alleles withdifferent CA-repeat numbers are reported to exist: 11, 13, 14 and 19(Ishimitsu, T et al. (2001) Peptides 22(11):1739-1744). The DNAvariation was reported not to affect transcription of the gene.

Study Design: Seventy (70) patients with refractory ovarian cancer wereenrolled in a Phase II clinical trial and treated with very low doescyclophosphamide (50 mg po in combination with Bevacizumab (10 mg/kg) IVevery 14 days. Different gene polymorphisms of angiogenic factors (VEGF+936 C/T, IL-8 −251 A/T, and AM 3′ less than (<) 14 CA repeats) wereexamined using PCR-RFLP. Primers are identified below.

Polymorph Forward Reverse Gene Location Function Primer Primer EnzymeVEGF936 C/T 936 reduced ACA CCA TCA TCG GTG N1a111 plasma levelsCCA TCG ACA ATT TAG for T allele GA CAG CAA GA IL8 −251 A/Tvariant higher TTG TTC TAA GGC AAA Mfe1 levels of IL8 CAC CTG CCACCT GAG CTC T TCA TCA CA AM 3′end CA associated AAG AGG CTG GCA ACArepeat with AGT CAG AAG TCA TTT TAA Hypertension GAT TGG TAT CCT GCA CAG

Results: Thirteen (13) patients had a partial response (PR (25%)) and 39were non responders (NR). Thirty-one patients had progressed. Medianfollow-up of 8.3 months with a median progression-free survival of 6.6months. Patients who were homozygous A/A or heterozygous A/T genotype atthe −251 locus in the IL-8 gene had a lower response rate than those whowere T/T (P=0.047 Fisher's exact test). Patients with VEGF (936) C/C hada median time to progression (TTP) of 6.5 months. Patients with with anyT (T/T, C/T) had a median TTP of 17.2 months. Patients carrying both AM3′end alleles with fewer than (<) 14 CA repeats had 3.4 months medianTTP, patients with at least one allele having at least (>) 14 CA 3′repeats showed a median TTP of 6.6 months; for patients having bothalleles with 14 or more (>) 3′ CA repeats, patients showed 8.7 months ofmedian TPP (P=0.0006 Log-rank test).

To the best of Applicant's knowledge, the data shows for the first timethat IL-8 (−251) is a potential molecular predictor of response toBevacizumab based chemotherapy. The data also show that both VEGF 936and the AM 3′ dinucleotide repeat polymorphisms are useful as molecularmarkers for time to tumor progression (TTP).

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. A method for determining whether a patient in need thereof will respond to anti-VEGF antibody based chemotherapy, comprising screening a suitable cell or tissue sample isolated from said patient for at least one genomic polymorphism or genotype selected from (i) IL-8(−251); (ii) VEGF(936); or (iii) AM (3′ CA repeats), wherein the patient is suitably treated if the corresponding genotype is (i) (T/T) for IL-8(−251); (ii) (T/T or C/T) for VEGF(936); or (iii) at least one AM allele having 14 or more 3′ CA repeats.
 2. The method of claim 1, wherein the anti-VEGF antibody based therapy comprises the administration of a Bevacizumab antibody or its biological equivalent.
 3. The method of claim 1, wherein the patient is a cancer patient suffering from a cancer of the group ovarian cancer, lung cancer, breast cancer and colon cancer.
 4. The method of claim 1, wherein the cancer treatment further comprises administration of an effective amount of cyclophosphamide.
 5. A method for selecting anti-VEGF antibody based chemotherapy for a patient in need thereof, comprising screening a suitable cell or tissue sample isolated from said patient for at least one genomic polymorphism or genotype selected from (i) IL-8(−251); (ii) VEGF(936); or (iii) AM (3′ CA repeats), wherein the therapy is selected if the patient's corresponding genotype is (i) (T/T) for IL-8(251); (ii) (T/T or C/T) for VEGF(936); or (iii) at least one AM allele having 14 or more 3′ CA repeats.
 6. The method of claim 5, wherein the anti-VEGF antibody based therapy comprises the administration of a Bevacizumab antibody or its biological equivalent.
 7. The method of claim 5, wherein the patient is a cancer patient suffering from a cancer of the group ovarian cancer, lung cancer, breast cancer and colon cancer.
 8. The method of claim 5, wherein the cancer treatment further comprises administration of an effective amount of cyclophosphamide.
 9. A method for treating a cancer patient selected from the group consisting of an ovarian cancer, a lung cancer, a breast cancer and a colon cancer, comprising: (a) screening a suitable cell or tissue sample isolated from said patient for at least one genomic polymorphism or genotype selected from (i) IL-8(−251); (ii) VEGF(936); or (iii) AM (3′ CA repeats), (b) identifying patients having a genotype selected from the group consisting of (i) (T/T) for IL-8(−251); (ii) (T/T or C/T) for VEGF(936); or (iii) at least one AM allele containing 14 or more 3′ CA repeats; and (c) administering an effective amount of an anti-VEGF antibody to the patient identified in step (b), thereby treating the patient.
 10. The method of claim 9, further comprising administering an effective amount of cyclophosphamide.
 11. The method of claim 9, wherein the anti-VEGF antibody is Bevacizumab or its biological equivalent. 